Effective immune responses to pathogens and vaccines critically depend on the formation of both short-term antibody secreting cells (ASC) and germinal centers. Despite the importance of generating both ASC and GCs, very little is known about the molecular changes that enable the initial divergence to these disparate fates during primary B cell divisions. Initial formation of germinal center B cells requires an elevated production of the transcriptional repressor Bcl6. Formation of ACS, on the other hand, involves a decrease in Pax5 followed by an increase of several other transcription factors including BLIMP-1. Bcl-6 and BLIMP-1 are known to antagonize each others expression, thus constraining and propelling cells along mutually exclusive pathways of differentiation. Although it is now well established that altered expression patterns of transcription factors mediates the shifts toward one lineage and away from what was previously a stable transcriptional program in naive B cells, it remains a mystery how the Pax5 dominated B cell program is interrupted in some cells and Bcl6 increased in others. Very little is know about the processes that occur during the multiple divisions preceding these more advanced stages of differentiation, despite their importance to the divergence of these pathways and GC establishment. Here we propose to investigate the progression of differentiation that precedes the appearance of known indicators of the GC or ASC transcriptional program. Using a validated strategy that sorts cells by the extent of cell division and expression of genes regulated by Bcl6 and Pax5, we will further characterize the cell subsets during their initial emergence and define the immediate precursors to lineage committed B cells. Aim 1: Define the progressive differentiation of B cells prior to lineage commitment in vivo. Antigen specific B cells will be assessed shortly after immunization for the emergence of transcription factor and phenotypic changes known to be associated with initial AFC and GC lineage formation. Expression levels of relevant molecules will be quantified by flow cytometry and/or qRT-PCR and correlates to cell division number and CD38, CD23 phenotype will be determined. RNA sequencing of B cell subsets sorted by cell division number and phenotype will identify transcriptional changes, including microRNA transcription, that immediately precede the earliest known molecular shifts in lineage development. Aim 2: Determine the heritage of lineage committed subsets through daughter cell analysis. To define the precursors of germinal center B cells, sorted subsets will be briefly cultured in vitro, allowed to complete the subsequent round(s) of division in vitro and assessed of their expression of Bcl6. Similarly sorted and fixed B cells, enriched for cells in the G2/M phase of the cell cycle, will be imaged via confocal microscopy directly ex vivo for extent of symmetry in the distribution of surface markers and transcription factors in conjoined daughters.